3HP has been produced by metabolically engineered strains of E. coli, see for instance WO2011/038364, EP2505656 and WO02/42418 and increased tolerance to 3HP thereby produced has been sought. Production of 3HP in yeast has also been described, see WO2012/019175.
3HP is toxic to S. cerevisiae and this limits the ability to produce 3HP using a metabolically engineered S. cerevisiae. It would be desirable to produce yeasts generally and S. cerevisiae strains especially that are resistant to the toxic effects of 3HP so as to provide a starting point for further genetic modification to provide an operative metabolic pathway for the production of 3HP. It would also be desirable to provide yeasts which do produce 3HP and which have enhanced resistance to its toxicity. Such 3HP tolerant yeasts can provide the basis for industrial production of 3HP by cultivation of yeasts.
The SFA1 gene (alternatively named ADH5) of Saccharomyces cerevisiae in its wild type form has the gene sequence of SEQ ID NO:1. The gene is thought to encode an S-(hydroxymethyl)glutathione dehydrogenase.
It has been reported that Sfa1p is a member of the class III alcohol dehydrogenases (EC: 1.1.1.284), which are bifunctional enzymes containing both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase activities. The glutathione-dependent formaldehyde dehydrogenase activity of Sfa1p is required for the detoxification of formaldehyde, and the alcohol dehydrogenase activity of Sfa1p can catalyze the final reactions in phenylalanine and tryptophan degradation. Sfa1p is also able to act as a hydroxymethylfurfural (HMF) reductase and catabolize HMF, a compound formed in the production of certain biofuels. Sfa1p has been localized to the cytoplasm and the mitochondria, and can act on a variety of substrates, including S-hydroxymethylglutathione, phenylacetaldehyde, indole acetaldehyde, octanol, 10-hydroxydecanoic acid, 12-hydroxydodecanoic acid, and S-nitrosoglutathione.
The five ethanol dehydrogenases (Adh1p, Adh2p, Adh3p, Adh4p, and Adh5p) as well as the bifunctional enzyme Sfa1p are also involved in the production of fusel alcohols during fermentation. Fusel alcohols are end products of amino acid catabolism (of valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and tyrosine) via the Ehrlich pathway and contribute to the flavour and aroma of yeast-fermented foods and beverages. They may also have physiological roles. For example, exposing cells to isoamyl alcohol, derived from catabolism of leucine, stimulates filamentous growth. Similarly, other fusel alcohols also stimulate filamentous growth in S. cerevisiae and biofilm formation in the pathogens Candida albicans and Candida dubliniensis. 
Transcription of SFA1 is controlled by Sko1p, a negative regulator of the Hog1p transcription regulation pathway. SFA1 is induced in sko1 null mutants and in cells overproducing the transcription factor Yap1p. Sfa1p expression is also induced by chemicals such as formaldehyde, ethanol and methyl methanesulfonate. sfa1 null mutants are viable and display hypersensitivity to formaldehyde, whereas overproduction of Sfa1p results in increased resistance to formaldehyde.
Sfa1p displays similarity to Adh1p, Adh2p, Adh3p and Adh5p, and to the alcohol dehydrogenases of Escherichia coli, Schizosaccharomyces pombe, Kluyveromyces marxianus, Kluyveromyces lactis, Candida albicans, Candida maltosa, horse, rat, and mouse, as well as human ADH2 and ADH3, which are associated with the development of Parkinson disease. Sfa1p also exhibits similarity to the glutathione-dependent formaldehyde dehydrogenase of Arabidopsis (FALDH), which is able to complement the formaldehyde-hypersensitivity defects of sfa1 null mutants. Sfa1p is also similar to the glutathione-dependent formaldehyde dehydrogenases of mouse and human (ADH5), which are involved in the catabolism of S-nitrosoglutathione, a type of S-nitrosothiol central to signal transduction and host defence.
Formaldehyde is formed by oxidative demethylation reactions in many plants and methylotrophic organisms, but Saccharomyces cerevisiae is a non-methylotrophic yeast and cannot metabolize methanol to formaldehyde. However, S. cerevisiae is exposed to exogenous formaldehyde from plant material or in polluted air and water.
Concentrations of formaldehyde of 1 mM or higher are cytostatic or cytotoxic to haploid wild-type cells. Any free formaldehyde in vivo spontaneously reacts with glutathione to form S-hydroxymethylglutathione. The level of enzymes involved in the degradation of formaldehyde, such as Sfa1p and Yjl068Cp, determine the level of formaldehyde toxicity, and cells overproducing Sfa1p are resistant to formaldehyde and null mutants in either sfa1 or yjl068c are hypersensitive to formaldehyde. Sfa1p is induced in response to chemicals such as formaldehyde (FA), ethanol and methyl methanesulphonate, and Yjl068Cp is also induced in response to chemical stresses. Molin and Blomberg, Molecular Microbiology (2006) 60(4), 925-938 reported that SFA1 overexpression enhanced formaldehyde tolerance in S. cerevisiae. They reported also that supplementation of a culture medium with glutathione restored DHA sensitivity of a gsh1Δ strain.
Formate dehydrogenase is encoded by FDH1/YOR388C and FDH2. In some strain backgrounds of S. cerevisiae, FDH2 is encoded by a continuous open reading frame comprised of YPL275W and YPL276W. However, in the systematic sequence of S288C, FDH2 is represented by these two separate open reading frames due to an in frame stop codon.
It has been reported that the effect of certain mutations in SFA1 or SFA1 deletion has been to decrease resistance to formaldehyde, S-nitrosoglutathione, and peroxynitrite (Fernández, et al, 1999).
There would on this basis appear to be no known reason why certain mutations in SFA1, or its overexpression should be expected to improve 3HP tolerance.
We have found that a genetic modification providing overexpression of SFA1 or providing certain mutations of SFA1 increases the ability of yeast and other cells to grow in the presence of normally inhibitory concentrations of 3HP. Furthermore, we have found that supplementation of a culture medium with glutathione also enables cells to grow in a normally inhibitory concentration of 3HP.
Cells incorporating such a genetic modification form an improved platform for further genetic engineering to provide a 3HP expression pathway in the cells.